Abstract The Bevilacqua lab has contributed to the field of RNA biology through two disparate approaches. One is rigorous ribozyme mechanism and the other is discovery-based RNA structural genomics. A major goal of this proposal is to bridge these two areas to discover new RNA biology and to characterize it at the molecular level. This proposal advances a set of testable hypotheses on RNA folding and catalysis, as well as proposes new technologies to enable discovery of novel RNA biology. Conservation of mechanistic strategies suggests ways in which small ribozyme self-cleavage is activated by two unique catalytic strategies. These strategies lead to specific changes in the hydrogen bonding status of the 2?OH that could lower its pKa to facilitate deprotonation. Kinetics experiments, pKa measurements, and calculations will be implemented and conducted on a diverse set of small ribozymes, as well as mechanistically related protein enzymes. In addition, cryoEM approaches to solve structures of small, unmodified ribozymes involving preparation of nano-objects structures and rapid vitrification will be pioneered. Techniques for cryoEM developed on small ribozymes will be applied to small RNA and RNP complexes to expand the impact of cryoEM on the RNA field. Evidence is provided for a third catalytic strategy, which is buffer catalysis in ribozymes, and a suite of experiments is proposed to test this. Buffer catalysis could help explain how diverse RNA enzymes work including large ribozymes. We also seek to understand RNA folding in vivo. Methods will be developed to measure RNA folding prediction rules under in vivo-like and in vivo conditions. Complex artificial cytoplasms will be developed and used to measure binding between a fluorescently labeled RNA and its unlabeled complement. Titrations in such `messy systems' will be accomplished on a qPCR plate reader as a function of temperature to provide van't Hoff parameters. Prediction rules for RNA folding will also be measured in vivo and genome-wide by applying Structure-seq to Bacillus subtilis growing at different temperatures. The prediction rules should improve RNA folding prediction under in vivo conditions. An inverse relationship between RNA and protein structure will be pursued by measuring ribosome profiling on select proteins. Overall, synergy between mechanistic and genomic approaches will be developed on multiple levels and includes pioneering techniques for detecting charged bases for mechanistic studies and applying them genome wide. In addition, weakly chelated in vivo-like Mg2+ conditions provide an ideal system for investigating stimulation of RNA catalysis and folding. Finally, RNA prediction rules developed in vivo will help describe the folding and function of catalytic RNAs. Computational approaches play a key role. They aid prediction of RNA structure from sequence with new prediction rules, help design cooperatively folding RNAs from structural descriptors, and allow testing of pKa lowering of the 2?OH nucleophile. By uniting the biophysical and genomics aspect of the lab, new discoveries in RNA biology will be made and understood at the molecular level.